09 Sep 2013: Report

How Tiny Fish Could Reveal Effects of Chemical Exposure

Researchers at a lab at Oregon State University are using zebrafish to assess the impacts of multiple chemical exposures. Their findings could help lead to a better understanding of how chemicals in the environment and in consumer products affect human health.

by elizabeth grossman

Inside Oregon State University’s Sinnhuber Aquatic Research Laboratory are rooms lined with tanks filled with thousands of zebrafish — silver, striped, one- to two-inch creatures often found in home aquariums. The lab is the domain of Robert Tanguay, professor of molecular toxicology at Oregon State, whose zebrafish are helping to answer one of the most pressing questions in environmental health and toxicology: What are the health effects of chemical mixtures?

Tanguay has pioneered the use of zebrafish in toxicology. His lab is on the leading edge of this research, designing new experimental techniques and equipment that are enabling scientists to assess the impacts of multiple chemical exposures. Using zebrafish, Tanguay and other scientists are zeroing in on why certain chemical components of crude oil spilled by the Exxon Valdez in 1989 continue to adversely affect fish survival in Alaska’s Prince William Sound. With zebrafish, they also are learning why pesticide runoff can impair Pacific Northwest salmon’s ability to navigate, and how

The lab is evaluating the toxicity of thousands of chemicals used in consumer products.

oil from the ruptured Deepwater Horizon well is affecting marine species’ health in the Gulf of Mexico.

But zebrafish research goes well beyond the impact of chemical exposures on marine life. Zebrafish studies are also being used to assess the potential human health impacts of the
chemical stew in U.S. Superfund sites. Tanguay’s lab is conducting research for the U.S. Environmental Protection Agency (EPA), evaluating the toxicity of thousands of chemicals used in countless consumer products. Zebrafish are also proving key to advancing our understanding of how particular chemical compounds affect the expression of individual genes that maintain and influence virtually every body system.

“Some people are still skeptical about how a fish can model human health,” said Tanguay during a tour of his lab earlier this month. “But during early development we are more similar to fish than at any other time in life.” Genes are particularly susceptible to environmental chemicals in early development. What happens then can set the stage for health throughout the rest of life.

The work of Tanguay and colleagues on mixtures is particularly important because, historically, chemicals have been evaluated and regulated one at a time. In reality, however, we are exposed to multiple chemicals daily throughout our lives. Because we now know that environmental chemical

Tanks of thousands of zebrafish line the laboratory at Oregon State University.

exposure can profoundly influence the development of disease, understanding how mixtures may affect human health has taken on an urgency.

“We live in a chemical soup,” says Linda Birnbaum, director of the U.S. National Toxicology Program and the National Institutes of Environmental Health Science (NIEHS).

Zebrafish have been used since the 1990s for genetic modeling and screening, but it was Tanguay, while working as a post-doctoral fellow in 1995, who suggested they might be used in toxicology. Because they are vertebrates, these prolifically reproducing fish turn out to be an extremely useful model for understanding how cells in other vertebrates, including humans, respond to environmental chemicals. Zebrafish, which progress from a translucent egg to a recognizable baby fish in just 24 hours, are ideal for investigating how chemicals may affect biological processes at various stages of development.

Because zebrafish eggs develop outside the mother, they can be manipulated, and Tanguay’s lab has devised equipment and techniques for isolating the eggs to examine the effects of chemicals. In one lab, Tanguay showed me a petri dish containing translucent zebrafish eggs, no bigger than a poppy seed, and the thread-fine, glass needles used to extract genetic material. Tanguay’s researchers use that material to determine

What’s learned from these zebrafish can be used to inform human health research.

which base pair — a rung in the DNA double-helix ladder — is targeted by a particular chemical exposure.

The results of these perturbations will tell the scientists if the exposure prompts any physical or health abnormalities. The zebrafish genome has been sequenced, enabling scientists to identify genes associated with specific diseases. Because many zebrafish genes have human counterparts, what’s learned from these fish can be used to inform human health research.

By figuring out which chemical targets a specific genetic receptor, scientists can begin to connect chemical exposure to health effects. Zebrafish development is readily visible, enabling scientists to identify changes in a fish’s phenotype — its physical characteristics. The zebrafish’s rapid development and short generational cycle make it possible to pinpoint effects that could be difficult to see in other animals. After linking chemical and physical effects, scientists then work to connect the phenotype with the genetic mechanisms that prompt it — or, as Tanguay says, to “connect phenotype with genotype.”

Some of these changes may result in physical abnormalities. Zebrafish exposed to certain dioxins, for example, had deformed vertebrae and skulls. Dioxin exposure also impaired regeneration of wounded fins. Other changes may become apparent in neurological, cognitive, and behavioral testing. Still others may be exhibited as abnormalities in reproduction, altered developmental timing, or changes in how metabolism and other body systems function.

Zebrafish are instrumental in research to determine the effects of ‘cocktails of pollution’ on fish species.

Zebrafish are instrumental in the research that Nat Scholz, the ecotoxicology program manager at the National Oceanic and Atmospheric Administration’s Northwest Fisheries Science Center, is conducting to investigate the effects of what he calls “cocktails of pollution” on different fish species, including Pacific Northwest salmon and herring. Among the combinations of chemicals he’s studying are those of various fuel oil compounds, particularly polycyclic aromatic hydrocarbons (PAHs) and pesticides.

Using zebrafish, Scholz and his colleagues have discovered that certain PAHs adversely impact cardiovascular development, leading to a heart failure syndrome that can be lethal to fish larvae and reduce adult fish survival. The work with zebrafish has enabled the scientists to discover why fish that looked normal were dying. By examining how low levels of PAH exposure interfere with genes involved in heart development, they have begun to understand how oil from the Exxon Valdez and Deepwater Horizon spills has impaired the cardiovascular function of fish at a cellular level.

While the PAH exposure prompts changes early in development, these effects may not become apparent until later on, when defects — in how the heart processes potassium and calcium and in heart structure — reduce cardiac function and cause swimming problems. Experiments with zebrafish have helped deduce which enzymes and genetic receptors are affected by which hydrocarbon compounds, something that’s not been possible with fish in the wild.

Tanguay’s lab is also working with the EPA and NIEHS on an ongoing project known as Tox21, which is assessing more than 10,000 commercially produced chemicals for a wide number of health effects. Zebrafish are being used as part of what is called “high-throughput” screening — the rapid testing of lots of chemicals. The goal is to fill the many gaps in toxicity data that exist for these chemicals and identify which chemical compositions or concentrations prompt specific health effects. Results of this screening are expected later this year.

“We call this Robot Row,” said Tanguay, showing me the lab area devoted to robotic machines designed to precisely dose hundreds of zebrafish eggs and genetic material samples. Because zebrafish develop so quickly, lots of variables can be examined concurrently using these machines. Tanguay’s lab uses what look like tiny paint palettes with an array of 96 little wells,

The lab is working to assess the toxicity of molecules invented to clean up environmental contaminants.

each of which gets a zebrafish sample that is then dosed by a machine with a particular chemical at different concentrations. Some experimentally treated eggs are allowed to develop, while others are halted to examine impacts during embryonic development.

Tanguay is also working with Terry Collins, professor of chemistry and director of Carnegie Mellon University’s Institute for Green Science, to assess the toxicity of molecules Collins has invented to clean up environmental contaminants. These compounds, known as TAML activators, can be used to remove dyes and other toxic chemicals from water. The TAML compounds appear to be successful in degrading these hazardous chemicals, but Collins wants to make sure their use doesn’t produce other harmful compounds or effects. Collins’ and Tanguay’s labs have been testing TAML activators on zebrafish to determine any potential toxicity, including on endocrine hormones.

The scientists are examining a complex range of possible outcomes that may result from the chemicals’ interaction with specific genetic receptors at different stages of development. If no abnormal effects are seen after the various TAML exposures, Collins will be closer to determining the safety of his chemical products. If adverse effects are seen, he will have the information he needs to begin redesigning his compounds so they are safe to use.

Tanguay’s lab has also designed equipment to test cognitive and behavioral responses in zebrafish. Similar to tests used with other lab animals, including humans, these experiments reveal how the brain, nervous system, and muscles may be affected by certain chemicals, such as organophosphate pesticides, known neurotoxicants that have been linked to adverse neurological effects in children. By using zebrafish, scientists have begun to locate which neurons these chemicals may affect at particular stages of development. These experiments are also being used to test the effects on fish of pesticide-laden water running off fields and roads.

The ultimate goal is to learn which chemical combinations, concentrations, and structures alter the normal function of genes and hormones. There is strong evidence, for example, that interference with endocrine system hormones and the genes that regulate them can lead to metabolic disorders such as diabetes and obesity. Research has also shown that endocrine-disrupting chemicals can upset reproductive, developmental, immune system, and neurological health. In addition, scientists have discovered that altered gene expression — distinctly different from genetic mutation — can be passed from one generation to another in what are called epigenetic effects. Pinpointing these precursors to disease is key to protecting and improving human health.

The ability to connect specific chemicals in the environment with mechanisms of disease is essential to finding and “eliminating those that are nasty,” explains Tanguay. In a world where manufactured chemicals are ubiquitous, the tiny, fecund zebrafish is playing a big role in discovering how this chemical stew is affecting our health.

COMMENTS

It's about time that we start studying subtoxic combinations of thousands of chemicals. Scientists are loathe to do such experiments because of the difficulty of interpreting results, but I have always suspected there are big surprises that have been missed due to our insistence that all experiments be designed so that results can be teased apart.

What if 1,000 common chemicals are each combined at 1/10,000 of their LD50 (or some more sophisticated level) — what would be the result? I hope that these researchers at least try a few "hopelessly complex" mixtures. Perhaps that is in effect what the testing of water runoff from Superfund sites is doing?

Posted by
Roger W Faulkner
on 09 Sep 2013

I wonder if these results will be available and appropriate for reference with GHS hazardous material classification, now required under OSHA hazard communication. Ecotox is exempt under OSHA, but other countries require it, and possibly EPA will too.

Posted by
Casey Wallace
on 16 Sep 2013

I think that it is refreshing to see that someone is finally doing something about trying to uncover the hidden effects of the numerous harmful chemicals we are constantly pumping into our environment. Why should we be poisoning ourselves and the generations to come? Every artificial thing that we introduce to the environment does not belong there, so why are we continuously poisoning our home and food? It makes no sense to me.

Posted by
Molly Graham
on 17 Sep 2013

It's very interesting to see how the effects of multiple chemical exposures at different parts of a life cycle are being studied, rather than only focusing on one chemical or stage of development. However, studying these effects on the zebra fish can only provide so much information, and while we can't study these effects on humans, it wouldn't be a bad idea to try testing other fish and animals in the same way to see what results they give.

Posted by
Tom Flynn
on 17 Sep 2013

This is an interesting topic, and I am glad to see someone is taking an interest in finding out what the chemicals we are subjected to everyday really can do to us. I am curious if the science, and potential effects from chemicals, are still valid after the test subject passes the early stages of development? Can this information still be applied to humans even after the fish has passed early developmental stages?

Posted by
Kelly Beall
on 18 Sep 2013

This is a great stepping stone in determining just how dangerous our everyday use chemicals are. Studies like these show the urgent need to adopt the precautionary principle in relation to chemical releases into the manufacturing market.

Posted by
DLR
on 19 Sep 2013

This is an interesting article, and I think that it's great that they are learning the possible outcomes of chemical exposure through zebrafish. Which ultimately can help see the impacts that these chemicals can have on human health. Also the information regarding gene expression changes in early life is fascinating and invaluable.

Posted by
Mahmoud A
on 06 Oct 2013

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ABOUT THE AUTHORElizabeth Grossman is the author of Chasing Molecules: Poisonous Products, Human Health, and other books. Her work has appeared in Scientific American, Salon, The Washington Post, The Nation, Mother Jones, Grist, and other publications. In earlier articles for Yale e360, she reported on recent studies suggesting a possible link between prenatal exposure to pesticides and the mental abilities of children and explored how declining bee populations threaten global food security.MORE BY THIS AUTHOR

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